Tracheobronchomalacia (TBM) and Complete Tracheal Rings (CTR), are congenital conditions characterized by abnormal tracheal cartilage and muscle and associated with high morbidity and mortality. The etiologies of TBM and CTR are poorly understood, and current interventions are limited to invasive surgery and palliative care. The overarching goal of the present application is to define underlying molecular mechanisms of congenital diseases affecting trachea development. Our published studies demonstrated a critical role for respiratory endoderm in determining the dorsal-ventral organization of the tracheal mesenchyme where cartilage and smooth muscle developed. Endodermal deletion of Wls (Wntless), a cargo receptor necessary for secretion of Wnt ligands, results in lack of tracheal cartilage that is replaced by abnormally organized smooth muscle, resembling TBM pathology. Our RNA Sequencing analysis identified Notum, and Bmp4, as candidate Wnt target genes in tracheal mesenchyme that regulate cartilage and trachealis smooth muscle formation downstream of epithelial Wls- induced signaling. We hypothesize that Notum acts by suppressing canonical Wnt signaling while simultaneously promoting non-canonical Wnt and BMP signaling required for tracheal mesenchyme differentiation. Thus, Notum is at the core of a novel model of regulation for the output of Wnt signaling wherein epithelial Wnt ligands first promote Wnt/b-catenin signaling and then turn it off to allow for non-canonical Wnt activity. To test the central hypothesis, we will: SA1: To determine the mechanism by which Notum promotes tracheal cartilage formation. Using novel mouse model and ex vivo studies, we will determine the extent to which deletion or overexpression of Notum affects Wnt/b-catenin dependent and independent signaling activity, and the epistasis among Notum, Wnt5a, and Wnt-b-catenin to pattern cartilage. SA2: To define the mechanism by which Bmp4 and Wnt signaling interact to mediate cell fate of tracheal mesenchyme. Performing in vivo and ex vivo studies we will determine whether Wnt/b- catenin synergizes with BMP to promote cartilage by inducing Notum and Wnt5a/Ror2 expression; and whether Notum and Bmp4 cooperate to repress ectopic muscle in ventral tracheal mesenchyme. SA3: To test the hypothesis that epithelial Wnt signaling directs cytoskeletal organization of trachealis smooth muscle. We will examine whether 1) canonical Wnt ligands from the epithelia act directly to promote myoblast cytoskeletal organization by regulating myogenic gene expression; 2) epithelial Wnt ligands activate non-canonical mesenchymal Wnt signaling working via Ror2 to mediate myoblast cytoskeletal organization. The proposed studies will identify critical molecular signaling pathways that mediate tracheal morphogenesis and will shed light on the pathology of TBM and CTR. Thus, these studies will lay the foundation for better diagnosis and therapeutic management of congenital and structural defects of the trachea. !